Method for enhancing nutrient absorption with astragalosides

ABSTRACT

The present application relates to a method for enhancing absorption of a nutrient in a subject in need thereof with an effective amount of an isolated astragaloside compound.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. patent applicationSer. No. 11/426,029, filed Jun. 23, 2006, which claims the benefit ofthe priority pursuant to 35 U.S.C. §119(e) of U.S. Provisional PatentApplication No. 60/694,097, filed Jun. 23, 2005. The contents of theprior applications are incorporated herein by their entireties.

BACKGROUND OF THE INVENTION

From the study of the human digestive system, it has been found that ahuge variety of nutritional substances are obtained by breaking down anddigesting the food in the gastrointestinal tract. The gastrointestinaltract is an important route by which the food is digested and absorbed.With regard to absorption, the nutritional substances, such as glucose,amino acids, vitamins and other smaller molecules are absorbed along theentire tract, either by diffusion or by specialized transport processes.Instead of moving freely across the intestinal membrane to the bloodstream or lymph, most of these nutritional substances are transported bya tightly regulated mechanism. Based on current understanding in cellbiology and physiology, the nutritional substances are transportedacross the cells with specific transport proteins and channels anchoredon the cell membrane.

In the example of glucose transportation, almost all of the cells have acarrier-mediated mechanism for the transport of glucose from blood. Formost cells, this transport occurs by facilitated diffusion using one ormore of the glucose transporters (GLUT) in a family of facilitatedglucose transporters. In these cases, net glucose transport occurs as aresult of an inwardly directed chemical gradient for glucose. In a fewcell types (e.g. those of intestinal mucosa and renal proximal tubule),uptake of glucose from an extracellular solution can occur against agradient of glucose in a so-called active transport mechanism, therebypermitting net absorption of glucose from a tissue compartment whoseglucose concentration may be lower than that of the blood. There are twoways in which a flow of energy can be coupled to transporters. Theprimary active transport requires energy be provided by adenosinetriphophatase (ATPase). The secondary active transport provides energyfrom the flow of ions from an area of higher concentration to one oflower concentration.

According to the secondary active transport model described above, Na⁺binds to transport protein on the luminal side of the cell causingconformational change of the transport protein, which opens the bindingsite for glucose. Then, glucose binds to the transport protein. Thetransport protein that is bound with both Na⁺ and glucose is subjectedto further conformational change to allow entry of glucose and Na⁺ intothe cells. This active transport of glucose involves a direct physicalcoupling of flows of Na⁺ and glucose, with the energy of the processbeing derived from the inwardly directed gradient for Na⁺. Since thetransport event includes a net movement of charge (the cationic Na⁺ ionwith the non-electrolyte glucose), the driving force for this uptakeincludes both the chemical gradient for Na⁺ and the potential differenceacross the membrane. As the glucose gradually accumulates in the cell,it is subsequently transported out to the blood vessel via a glucoseconcentration gradient by facilitated diffusion. Similarly, othernutritional substances may be absorbed with the transport mechanismdescribed above.

Astragalus root (Radix Astragali) has been used as a traditional Chinesemedicine that mainly serves to invigorate the function of the spleen andincrease stamina and endurance. Astragalus root (Radix Astragali) wasfound to enhance the immune system and help the human body resist virusinfections, particularly in the lungs, by increasing production ofinterferon, an immune factor that inhibits viral growth. Astragalus roothas been used as an adjuvant therapy in the treatment of colds andinfluenza. Radix Astragali was also reported to have effects oncardiovascular activity. Alcohol extracts of Radix Astragali enhancedboth the contractility and contraction amplitude of isolated frog ortoad hearts. Furthermore, astragalosides isolated from Radix Astragalihave been reported to exert a positive inotropic effect on isolated rathearts.

However, Astragalus membranaceus var. mongholicus has not been impliedin regulating nutrient absorption and transportation. None of the studyor research has focused on regulating the nutrient absorption usingsaponin compounds purified from Chinese herbal medicines, particularlyAstragalus membranaceus var. mongholicus.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a method for enhancing the absorption ofa nutrient, such as glucose, an amino acid (e.g. arginine ortryptophan), and a vitamin (e.g., folate), in a subject in need thereof.This method includes the steps of identifying a subject who needsup-regulation of nutrient absorption and administering to the subject aneffective amount of an isolated astragaloside compound (e.g., fromAstragalus membranaceus var. mongholicus). “An effective amount” as usedherein refers to the amount of each active agent required to confertherapeutic effect on the subject, either alone or in combination withone or more other active agents. Effective amounts vary, as recognizedby those skilled in the art, depending on route of administration,excipient usage, and co-usage with other active agents. Subjects in needof this regulation include elderlies, juveniles, pregnant or menopausalwomen, post-surgery patients, and patients suffering from long-termpressure, abnormal metabolism (e.g., type II diabetics), a weakenedimmune system (e.g., leukemia patients, HIV carriers, and organtransplantation recipients), or other diseases/disorders listed in Table1 below.

The astragaloside compound, preferably isolated, is an cycloartanecompound of Formula (A):

wherein R₁ is selected from the group consisting of H, OH, O-acetyl,O-xylopyranosyl, O-(2-acetylxylopyranosyl), O-(3-acetylxylopyranosyl),O-(2,3-diacetylxylopyranosyl), O-(2,4-diacetylxylopyranosyl),O-xylopyranosyl-(1-2)-β-D-glucopyranosyl andO-xylopyranosyl-(1-2)-α-arabinopyranosyl; R₂ is selected from the groupconsisting of H, OH, O-acetyl and O-glucopyranosyl, O-xylopyranosyl; R₃is selected from the group consisting of H, OH and O-acetyl; and R₄ isselected from the group consisting of

The term “isolated astragaloside compound” used herein refers to anastragaloside compound prepared by a synthetic method or enriched from anatural source (e.g., Astragalus membranaceus var. mongholicus). Forexample, an isolated astragaloside compound is a preparation thatcontains at least 40% (e.g., at least 95%) of the astragaloside compoundby dry weight. Purity of an isolated compound can be measured by, e.g.,column chromatography, mass spectrometry, high performance liquidchromatography (HPLC), NMR, or any other suitable methods.

Preferably, the astragaloside compound used in the method of thisinvention is selected from the group consisting of astragaloside I ofFormula I:

astragaloside II of Formula II:

astragaloside III of Formula III:

astragaloside IV of Formula IV:

isoastragaloside I of Formula V:

astragaloside VI of Formula VI:

isoastragaloside II of Formula VII:

and cycloastragenol-6-O-β-D-glucopyranose of Formula VIII:

Additional features and advantages of the present invention will be setforth in part in the description which follows, and in part will beapparent from the description, or may be learned by practice of theinvention. The features and advantages of the invention will be realizedand attained by means of the elements and combinations as described.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. For the purpose of illustrating the invention,there are shown in the drawings embodiments, which are presentlypreferred. It should be understood, however, that the invention is notlimited to the precise arrangements and instrumentalities shown.

In the drawings:

FIG. 1 is a line graph showing the glucose uptake rates of the Caco2cells treated with the isolated astragaloside AS4 of Formula IV ofselected concentrations;

FIG. 2 is a line graph showing the arginine absorption rates measured inthe Sink-transport across to basolateral chambers when the Caco2monolayers were treated with the isolated astragaloside AS1 of Formula Iof selected concentrations;

FIG. 3 is a line graph showing the tryptophan absorption rates measuredin the Sink-transport across to basolateral chambers when the Caco2monolayers were treated with the isolated astragaloside AS1 of Formula Iof selected concentrations;

FIG. 4 is a line graph showing the folate uptake rates of the Caco2cells treated with the isolated astragaloside AS1 of Formula I of aselected concentration.

DETAILED DESCRIPTION OF THE INVENTION

To better understand the present invention, the terms used herein areexplained in further detail. An astragaloside is defined as a triterpenesaponin compound extracted from Radix Astragali, the dried root ofAstragalus membranaceus (Fisch) Bunge and Astragalus mongholicus Bunge(Fabaceae).

As used herein, the singular forms “a”, “an”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a compound” includes a plurality of suchcompounds.

The term “absorption” as used herein refers to uptake of a nutrient viaa passage through the intestinal epithelium and into the blood or lymph.

The term “gut cells” as used herein generally include enterocytes,mucosal cell, and cells of intestinal epithelium responsible fornutrient absorption of the body.

The term “subject” as used herein refers to any animal, preferablyincluding humans, where absorption of nutrients occurs across gut cellsin the subject's gastrointestinal tract.

The present invention is based at least in part on the unexpecteddiscovery that a number of astragaloside compounds enhancetransportation of certain nutrients across a monolayer of the gut cellslining the gastrointestinal tract. See Examples 1-4 below. Thus, thisinvention provides a method for up-regulating the absorption of anutrient with a astragaloside compound in a subject in need thereof.Table 1 below provides examples of the particular types of subjects whoneed enhanced absorption of particular nutrients:

TABLE 1 Subjects Who Need Up-Regulation of Absorption of CertainNutrients Nutrient Subject In Need Glucose Elderlies, athletes,alcoholics, juveniles, post-surgery patients, malnutrition patients, andpatients having digestive tract disorders Arginine Juveniles, athletes,over-weight patients, patients suffering from cardiovascular disease, aweakened immune system, physical injury (e.g., burn trauma), anderectile dysfunction Tryptophan Over-weight patients, patients sufferingfrom insomnia, a weakened immune system, and long-term pressure FolateElderlies, pregnant women, nursing mothers, and patients suffering frominsomnia, depression, cardiovascular disease, or long-term pressure

The astragaloside compounds may be formulated into tablets, pills,capsules, liquid formulations and powder to be orally administered tothe individual with nutrient absorption problem or mal-absorptionsyndrome, which is an alteration in the ability of the intestine toabsorb nutrients adequately into the bloodstream. In one embodiment ofthe preparation of the liquid formulation, one or more of theastragaloside compounds may be dissolved in any solvent, preferably in aco-solvent, to produce a liquid formulation of the astragalosidecompounds (such as, 10 mg of any of the astragaloside compounds may bedissolved in one mL of Transcutol® P [2-(2-ethoxyethoxy)ethanol]). Also,the astragaloside compounds may be optionally mixed with other nutrientfactors, additives, stabilizing agents, carriers, binders and fillers toproduce dietary supplements, beverages, food, and animal feeds for asubject in need of enhanced nutrient absorption. It may be apparent toone skilled in the art in view of the present disclosure to administerthe astragaloside compounds in combination or in a cocktail manner withother ginsenosides and astragalosides to provide a synergistic oraccumulative effect on the nutrient absorption.

The astragaloside compounds may be prepared by any standard methodologyor known methods or knowledge in the art. According to the invention,the isolated astragaloside compounds, e.g., from Astragalus membranaceusvar. mongholicus, include the astragalosides. They may be isolated byother available extraction and isolation methods known to those skilledin the art. For example, the isolated astragaloside compounds may alsobe enriched from other Chinese herbal plants or vegetation to providethe same regulatory effect on nutrient absorption function. According toan embodiment of the invention, the astragaloside compounds may beobtained by a method comprising the steps of grinding the root ofAstragalus membranaceus var. mongholicus and extracting with alcohol toproduce an alcohol extract. The alcohol extract of Astragalusmembranaceus var. mongholicus may be separated and purified to giveseven known cycloartane compounds including astragaloside I (hereinafter“AS1”) of Formula I:

astragaloside II (hereinafter “AS2”) of Formula II:

astragaloside III (hereinafter “AS3”) of Formula III:

astragaloside IV (hereinafter “AS4”) of Formula IV:

isoastragaloside I (hereinafter as IsoAS1) of Formula V:

astragaloside VI (hereinafter “AS6”) of Formula VI:

and isoastragaloside II (hereinafter as IsoAS2) of Formula VII:

The cycloartane compounds may be separated and purified with silica geland reversed phase chromatography. AS4 may further be hydrolyzed usingnaringinase to obtain a metabolite, such as cycloastragenol6-O-β-D-gluco-pyranose (hereinafter “AA”) of Formula VIII:

In one embodiment, the absorption of glucose is enhanced by facilitatingthe transportation of glucose across the gut cells of the subject inneed of the absoption enhancement with administration of one or more ofthe above-discribed astragaloside compounds at a concentration of about0.001 μM to about 5 μM; wherein the astragaloside compound is AS1 ofFormula I, AS4 of Formula IV, AS6 of Formula VI, IsoAS2 of Formula VII,or AA of Formula VIII.

In another embodiment, the absorption of arginine is enhanced byfacilitating the transportation of arginine across the gut cells of thesubject in need of the absorption enhancement with administration of oneor more of the above-discribed astragaloside compounds at aconcentration of about 0.001 μM to about 5 μM; wherein the astragalosidecompound is AS1 of Formula I, AS2 of Formula II, AS3 of Formula III, AS4of Formula IV, IsoAS1 of Formula V, AS6 of Formula VI, IsoAS2 of FormulaVII, or AA of Formula VIII.

In still another embodiment, the absorption of tryptophan is enhanced byfacilitating transportation of tryptophan across the gut cells of thesubject in need of the absorption enhancement with administration of oneor more of the above-discribed astragaloside compounds at aconcentration of about 0.001 to about 5 μM; wherein the astragalosidecompound is AS1 of Formula I, AS2 of Formula II, AS3 of Formula III, AS4of Formula IV, IsoAS1 of Formula V, AS6 of Formula VI, IsoAS2 of FormulaVII, or AA of Formula VIII.

In yet another embodiment, the absorption of folate is enhanced byfacilitating transportation of folate across the gut cells of thesubject in need of the absorption enhancement with administration of oneor more of the above-discribed astragaloside compounds at aconcentration of about 0.001 μM to about 5 μM; wherein the astragalosidecompound is AS1 of Formula I, AS2 of Formula II, AS3 of Formula III, AS4of Formula IV, IsoAS1 of Formula V, AS6 of Formula VI, IsoAS2 of FormulaVII, or AA of Formula VIII.

The present invention is more specifically explained by the followingexamples. However, it should be noted that the present invention is notlimited to these examples in any manner.

Example 1 Regulatory Effects of Isolated Astragalosides on GlucoseUptake Cell Culture

To evaluate the effect of the isolated astragaloside compound on theuptake of nutrient substances across the intestinal lumen, Caco-2 cellswere grown on permeable filter as an experimental model. Caco2 cellsoriginate from human colonic adenocarcinoma and spontaneouslydifferentiate into an enterocyte-like phenotype after two weeks. TheCaco-2 cell line, derived from a human colorectal carcinoma, has beenused as an in vitro model system for studying drug absorption ingastrointestinal tract. These cells form monolayers with well-developedtight-junctions, and have been evaluated in details as an in vitro modelto study both transcellular transport of nutrients and drugs inintestinal lumen.

Caco-2 cells were obtained from the ATCC (American Type CultureCollection). The cells were maintained in Dulbecco's modified Eaglemedium (DMEM) containing 4.5 g/L glucose and 25 mM Hepes, supplementedwith 10% fetal calf serum, 100 U/mL penicillin G and 10 μg/Lstreptomycin. The medium was changed every second day. The cells wereroutinely checked for Mycoplasma in monthly intervals. Caco-2 cells werecultured on semi-permeable membranes to differentiate into a highlyfunctionalized epithelial barrier with remarkable morphological andbiochemical similarity to the small intestinal columnar epithelium. TheCaco-2 cell monolayers could therefore be used to study the membranetransport properties of many compounds. To trypsinize the cells, theculture dish was washed once with phosphate-buffered saline (PBS)followed by adding trypsine-EDTA for 10 minutes. The trypsinized cellswere separated and filtered into single cells using a 35-μm strainer cap(Falcon 2235) before being seeded for further experiments.

Cell Viability Assay

To investigate whether the isolated astragalosides were toxic to theCaco2 cells, cell viability assay was carried out using culture mediumsupplemented with 1% and 10% FBS, respectively. The cells were seeded ata concentration of 5000 cells/well in a 96-well plate. To eliminate theboundary effect of the cell growth, the cells were only seeded in 60wells of the middle area of the plate, whereas 36 wells at thesurrounding area of the plate were filled only with 100 μL of PBS. Oncethe cells were attached to the plate, the cells were incubated in mediumcontaining the isolated astragalosides at various doses (0, 1, 10, 20and 50 μM). After 3 days, the culture medium was replaced with freshmedium containing the same compounds and incubated for 2 more daysbefore the cells were assayed for cell viability.

The cell viability was determined by a Cell Counting Kit-8 (CCK-8,Dojindo Laboratories, Kumamoto, Japan) assay that is based on redoxreaction of NADH in the living cells with cell proliferation reagentWST-8. WST-8 was reduced by dehydrogenases in electron transport chain(ETC) of mitochondria in the cells to give a yellow-colored formazanproduct, which was soluble in the tissue culture medium. The amount offormazan dye generated by the activity of dehydrogenases in the cellswas directly proportional to the number of the living cells. Therefore,a greater light absorbance detected by ELISA reader at wavelength of 450nm indicated presence of a larger number of the living cells.

The CCK-8 assay was carried out by adding 10 μL of the CCK-8 reagent ineach well of 96-format plate. The plate was then covered with aluminumfoil and further incubated for two hours before measuring for absorbanceat wavelengths of 450 nm by using an ELISA reader.

Glucose Uptake Assay

Caco-2 cells (5×10⁴) were seeded in a 48-well plate and maintained inculture medium (DMEM with 10% FBS, 1% nonessential amino acids,L-glutamine, penicillin G (100 U/mL), streptomycin (10 μg/mL), andamphotericin B (2.5 μg/mL) in a 37° C. incubator for 10 days for thecells to differentiate. The culture medium was changed once every twodays. The cells were then washed with PBS before replenishing with theculture medium containing 5% FBS and various astragalosides at theindicated concentrations (0.01, 0.1 and 1 μM) for 48 hours. The Caco2cells were washed out of remaining glucose with PBS and replaced in theglucose buffer (80 mM NaCl, 100 mM mannitol, 20 mM Tris-HCl, pH 7.4, 3mM K₂HPO₄, 1 mM CaCl₂, 1 mg/mL BSA) for 1 hour. Glucose uptake wasinitiated by replacing the glucose buffer with 0.2 ml of glucose buffercontaining 2 μCi/mL of ¹⁴C-glucose and unlabeled cold glucose to give afinal glucose concentration of 25 mM. Glucose uptake was stopped byremoving the glucose buffer and washing with PBS at designated timeintervals. The cells were lysed in 0.2 mL of 0.2 N NaOH, and 20 μL ofthe cell lysate were transferred to the filter-bottomed UniFilter plates(Perkin-Elmer, Wellesley, Mass., USA) and dried in a vacuum oven at 37°C. The bottom of the UniFilter plate was sealed and 25 μL of thecounting solution were added into each well. Adhesive plate sealers wereused in place of the lids and radioactivity of each sample was countedusing the microplate liquid scintillation counter (TopCount, PackardNXT, Packard BioScience Company, Meriden, Conn., USA). The amount ofglucose accumulated in the cells was calculated and normalized toprotein concentration, and uptake rate was expressed as nanomoles ofglucose per minutes per milligram of cell protein (nmol/min/mg). Proteinconcentration was determined by a standard Bicinchoninic acid (BCA)protein assay. Nonspecific glucose uptake was measured by adding 2 μCiof L-[¹⁴C]-glucose and subtracting from each determination to obtainspecific glucose uptake.

In the cell viability assay, the isolated astragaloside did notgenerally affect growth of Caco2 cells at a concentration range from 1to 50 μM except when the AS1 of Formula I at a concentration of 10 μMwas administered to Caco2 cells. Therefore, the isolated astragalosidewas administered in the subsequent glucose uptake test or folate uptaketest at a concentration range that did not cause cell toxicity.Preferably, the isolated astragaloside was administered at aconcentration range of about 0.001 μM to about 1 μM.

From the glucose uptake assay shown in Table 2, it was found thatisolated astragalosides, such as AS1 of Formula I, AS4 of Formula IV,AS6 of Formula VI, AA of Formula VIII, and IsoAS2 of Formula VII hadregulatory effects on the glucose uptake of the Caco2 cells. The amountof glucose uptake was determined and expressed as “nmoles for each mg ofcell protein.” As shown in FIG. 1, the Caco2 cells treated with AS4 ofFormula IV showed a much higher glucose uptake rate than the controlgroup. The regulatory effects of the isolated astragalosides on theglucose transport in Caco2 cells are listed in Table 2 below, whereinthe arrows that point up represent the enhancing effect on the glucoseuptake.

TABLE 2 Regulatory effects of astragalosides on glucose uptake Uptakerate Compound (μM) (nmol/mg/min) Percentage (%) * Control 6.3720 ±1.9290 100 — AS1 1 6.2780 ± 1.9930 98.52 0.1 7.8260 ± 1.7510 122.82 ↑0.01 9.3510 ± 1.1370 146.75 ↑ AS4 1 9.0020 ± 1.8300 141.27 ↑ 0.1 10.460± 2.6690 164.16 ↑ 0.01 11.670 ± 2.5800 183.15 ↑ AS6 1 7.8050 ± 1.1830122.49 ↑ 0.1 7.0070 ± 1.9470 109.97 ↑ 0.01 7.6360 ± 1.7330 119.84 ↑ AA 19.2830 ± 2.1150 145.68 ↑ 0.1 9.3460 ± 2.3210 146.67 ↑ 0.01 11.450 ±3.2760 179.69 ↑ IsoAS2 1 6.1520 ± 2.4610 96.55 0.1 6.8210 ± 1.6630107.05 ↑ 0.01 7.3720 ± 2.5700 115.69 ↑

It is concluded that the absorption of glucose can be enhanced by theastragaloside isolated from Astragalus membranaceus var. mongholicus,including AS1 of Formula I, AS4 of Formula IV, AS6 of Formula VI, AA ofFormula VIII or IsoAS2 of Formula VII.

Example 2 Regulatory Effect of Isolated Astragalosides on ArginineAbsorption Arginine Absorption Assay

In measuring transport of arginine across the Caco-2 cell monolayer,both sides of the transwells were washed with arginine incubation bufferconsisting of: 137 mM NaCl, 10 mM Hepes, 0.3 mM NaH₂PO₄, 0.3 mM K₂HPO₄,5.4 mM KCl, 2.8 mM CaCl₂, 1 mM MgSO₄, 10 mM glucose, adjusted to pH 7.4.Then, the cell layer was preincubated in the incubation buffer at 37° C.for 1 h. The volume of incubation buffer was 0.2 mL and 0.9 mL in theapical and basolateral chambers, respectively. The cells were replacedwith fresh incubation medium in both chambers prior to the transportexperiment. The transport experiment was initiated by replacing theincubation solution on the apical side with solution containing 10 mM ofL-arginine in which 0.125 μCi/mL of L-[³H]-arginine was included. Atdesignated time intervals, 10 μL-solution samples were removed from thebasolateral side and radioactivity of each sample was counted using amicroplate liquid scintillation counter (TopCount, Packard NXT). Duringthe experiment, when a 10 μL-solution sample was removed from thebasolateral side every time, 10 μL buffer was supplemented to keep thevolume constant. The uptake of [³H]-mannitol was used to correct fornonspecific transport of molecules across the monolayer membrane.Results were expressed as the nanomoles of arginine transport across theCaco-2 cell monolayers with respect to time in minutes (nmol/min).

From the arginine absorption assay results shown in Table 3, it wasfound that isolated astragalosides, such as AS1 of Formula I, AS2 ofFormula II, AS3 of Formula III, AS4 of Formula IV, AS6 of Formula VI, AAof Formula VIII, IsoAS1 of Formula V and IsoAS2 of Formula VII hadregulatory effects on the arginine transport across the Caco2 cellmonolayer. Referring to FIG. 2 and Table 3, the arginine transport ratewas increased when the Caco2 cell monolayer was treated with AS1 ofFormula I or AS2 of Formula II at a concentration from 0.001 μM to 0.1μM. The arginine transport rate was increased when the Caco2 cellmonolayer was treated with AS3 of Formula III, AS4 of Formula IV, AS6 ofFormula VI, AA of Formula VIII, IsoAS1 of Formula V or IsoAS2 of FormulaVII, respectively, at a concentration from 0.01 μM to 1 μM. Theregulatory effects of the isolated astragalosides on the argininetransport in Caco2 cells are listed in Table 3 below, wherein the arrowsthat point up represent the enhancing effect on the arginine transport.

TABLE 3 Regulatory effects of isolated astragalosides on Argininetransport Compound (μM) Transport rate (nmol/min) Percentage (%) *Control 10.6855 ± 0.2523 100 — AS1 0.1 15.7300 ± 1.1250 147.21 ↑ 0.0116.2324 ± 0.7215 151.91 ↑ 0.001 14.2554 ± 0.5851 133.37 ↑ AS2 0.117.2771 ± 1.6170 161.69 ↑ 0.01 16.2358 ± 1.6190 151.94 ↑ 0.001 14.6355 ±1.2910 136.97 ↑ AS3 1 15.8341 ± 1.0000 148.18 ↑ 0.1 13.2858 ± 1.4110124.33 ↑ 0.01 13.0084 ± 1.0510 121.74 ↑ AS4 1 17.2241 ± 0.3759 161.19 ↑0.1 18.4575 ± 0.5955 172.73 ↑ 0.01 16.7245 ± 0.2890 156.52 ↑ AS6 113.5942 ± 1.2760 127.22 ↑ 0.1 14.9986 ± 1.3200 140.36 ↑ 0.01 13.9283 ±1.7330 130.35 ↑ AA 1 17.3164 ± 1.6150 162.06 ↑ 0.1 18.2169 ± 1.8700170.48 ↑ 0.01 21.3347 ± 1.7800 199.66 ↑ IsoAS1 1 14.4734 ± 1.1350 135.41↑ 0.1 21.3107 ± 1.5130 199.44 ↑ 0.01 14.4776 ± 0.5519 135.49 ↑ IsoAS2 112.6518 ± 0.3680 118.40 ↑ 0.1 14.1059 ± 0.1815 132.01 ↑ 0.01 14.7577 ±0.2837 138.11 ↑

It is concluded that the absorption of arginine can be enhanced with theadministration of astragalosides isolated from Astragalus membranaceusvar. mongholicus, including AS1 of Formula I, AS2 of Formula II, AS3 ofFormula III, AS4 of Formula IV, AS6 of Formula VI, AA of Formula VIII,IsoAS1 of Formula V or IsoAS2 of Formula VII.

Example 3 Regulatory Effects of Isolated Astragalosides on TryptophanAbsorption Trytophan Absorption Assay

The experimental procedures similar to those in Example 2 were used formeasuring the uptake of tryptophan molecules across the Caco-2 membrane,except using a tryptophan incubation buffer consisting of 137 mM cholinechloride, 10 mM Hepes, 0.6 mM KH₂PO₄, 5.4 mM KCl, 2.8 mM CaCl₂, 1 mMMgSO₄, and 10 mM glucose, and having its pH adjusted to 7.4. Resultswere expressed as the nanomoles of tryptophan transport across theCaco-2 cell monolayers with respect to time in minutes (nmol/min).

From the tryptophan absorption assay results shown in Table 4, it wasfound that isolated astragalosides, such as AS1 of Formula I, AS2 ofFormula II, AS3 of Formula III, AS4 of Formula IV, AS6 of Formula VI, AAof Formula VIII, IsoAS1 of Formula V and IsoAS2 of Formula VII hadregulatory effects on the tryptophan transport across the Caco2 cellmonolayer. As shown in FIG. 3 and Table 4, the tryptophan transport ratewas increased when the Caco2 cell monolayer was treated with AS2 ofFormula II, AS3 of Formula III, AS4 of Formula IV, AS6 of Formula VI, AAof Formula VIII, IsoAS1 of Formula V or IsoAS2 of Formula VIIrespectively at a concentration from 0.01 μM to 1 mM, and with AS1 ofFormula I at a concentration from 0.01 μM to 0.1 mM. The regulatoryeffects of the isolated astragalosides on the tryptophan transport inCaco2 cells are listed in Table 4 below, wherein the arrows that pointup represent the enhancing effect on the tryptophan transport.

TABLE 4 Regulatory effects of isolated astragalosides on Tryptophantransport Compound (μM) Transport rate (nmol/min) Percentage (%) *Control 8.9420 ± 0.3670 100 — AS1 1 16.000 ± 1.3190 178.93 ↑ 0.1 23.130± 1.3120 258.67 ↑ 0.01 22.220 ± 0.8695 248.49 ↑ AS2 1 — — — 0.1 11.650 ±0.5789 130.28 ↑ 0.01 10.290 ± 0.4115 115.07 ↑ AS3 1 24.200 ± 1.0260270.63 ↑ 0.1 13.590 ± 1.0080 151.98 ↑ 0.01 14.290 ± 1.3910 159.81 ↑ AS41 9.6640 ± 0.2770 108.07 ↑ 0.1 12.730 ± 0.4470 142.36 ↑ 0.01 10.130 ±0.7025 113.29 ↑ AS6 1 15.490 ± 0.2161 173.23 ↑ 0.1 13.850 ± 0.6567151.87 ↑ 0.01 15.510 ± 0.3688 173.45 ↑ AA 1 12.100 ± 0.5197 135.32 ↑ 0.114.000 ± 0.6445 156.56 ↑ 0.01 11.900 ± 0.6231 133.08 ↑ IsoAS1 1 10.600 ±0.8058 118.54 ↑ 0.1 13.370 ± 0.3301 149.52 ↑ 0.01 10.370 ± 0.8808 115.97↑ IsoAS2 1 20.070 ± 0.1931 224.45 ↑ 0.1 13.060 ± 0.5530 146.05 ↑ 0.0113.000 ± 0.5547 145.38 ↑

It is concluded that the absorption of tryptophan may be enhanced withthe administration of the astragaloside isolated from Astragalusmembranaceus var. mongholicus, including AS1 of Formula I, AS2 ofFormula II, AS3 of Formula III, AS4 of Formula IV, AS6 of Formula VI, AAof Formula VIII, IsoAS1 of Formula V or IsoAS2 of Formula VII.

Example 4 Regulatory Effects of Isolated Astragalosides on Folate UptakeFolate Uptake Assay

The Caco2 cells were subjected to folate uptake test in a manner similarto that described in the glucose uptake assay in Example 1 above. In thefolate uptake test, the Caco2 cells were pretreated with the culturemedium containing 5% FBS and isolated astragalosides at a concentrationof 0.1 μM for 2 days before the cells were cultured in a folate uptakebuffer (Hank's balanced salt solution, supplemented with 0.14 g/L CaCl₂,0.1 g/L MgCl₂, and 0.1 g/L MgSO₄, pH 6.0) for 1 hour. The buffer wasthen aspirated, and uptake was initiated by adding 0.2 mL of freshfolate uptake buffer containing 2 μCi/mL radioactive folate(3,5,7,9-³H-folic acid, 25 mCi/mmol, ARC) and cold, unlabeled folategiving a final folate concentration of 5 μM. The folate uptake wasterminated by removing the uptake buffer at designated time intervals.The cells were then washed three times with ice-cold PBS and lysed bythe addition of 0.2 mL of 0.2N NaOH, followed by incubation at 65° C.for 20 min. Intracellular uptake of ³H-folate was determined bytransferring 20 μL of the cell lysate to the filter-bottomed UniFilterplates (Perkin-Elmer) and counting as described previously in Example 1.The amount of folate accumulated in the cells was calculated andnormalized to protein concentration, and uptake rate was expressed aspicomoles of folate per minutes per milligram of cell protein(pmol/min/mg). Protein concentration was determined by a standardBicinchoninic acid (BCA) protein assay as described above.

Referring to FIG. 4, Caco2 cells treated with AS1 of Formula I at theconcentration of 0.1 μM was found to exhibit an increased folate uptakefrom the control group having non-treated Caco2 cells. The regulatoryeffects of the isolated astragalosides on the folate uptake in Caco2cells are listed in Table 5 below, wherein the arrows that point uprepresent the enhancing effect on folate uptake.

TABLE 5 Regulatory effects of isolated astragalosides on folate uptakeUptake rate (pmol/mg/min) Percentage (%) * Control 53.140 ± 3.5540 — 0.1μM AS1 79.710 ± 3.0410 150.00 ↑ Control 54.220 ± 3.1730 — 0.1 μM AS269.970 ± 3.7720 129.05 ↑ Control 55.280 ± 0.8527 — 0.1 μM AS3 80.380 ±6.2170 145.41 ↑ Control 56.030 ± 0.9678 — 0.1 μM AS4 75.710 ± 5.2390135.12 ↑ Control 60.240 ± 6.6510 — 0.1 μM AS6 84.560 ± 4.7200 140.37 ↑Control 53.010 ± 6.3290 — 0.1 μM AA 84.030 ± 4.9410 158.52 ↑ Control50.720 ± 3.7550 — 0.1 μM 73.460 ± 3.6060 144.83 ↑ IsoAS1 Control 53.19 ±1.98  — 0.1 μM 86.63 ± 2.82  162.87 ↑ IsoAS2

It is concluded that the uptake of folate can be enhanced with theadministration of the astragaloside isolated from Astragalusmembranaceus var. mongholicus, including AS1 of Formula I, AS2 ofFormula II, AS3 of Formula III, AS4 of Formula IV, AS6 of Formula VI, AAof Formula VIII, IsoAS1 of Formula V or IsoAS2 of Formula VII.

Although the above examples described regulating nutrient absorption ofthe colon cancer cells, it should be noted that the present invention isnot limited as such. The gut cells and cells of gastrointestinal systemshould also be expected to benefit from the regulatory effect of theastragaloside compounds proposed in the present invention as long asthese cells have similar nutrient transporting mechanisms. Besides aregulatory role in glucose, arginine, tryptophan and folate absorption,the astragaloside compounds described in the present invention mayequivalently apply to regulate absorption of nutrients which includevitamins, amino acids, hormones, growth factors, and other elementsimportant for cell metabolism. Moreover, the nutrient absorption testand nutrient uptake test described in the embodiments may be implementedinterchangeably for assessing and evaluating the regulatory effect ofthe isolated astragaloside on the nutrient absorption of the individualaccording to the present invention.

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications within the spirit and scope of thepresent invention as defined by the appended claims.

1-27. (canceled)
 28. A method for enhancing absorption of arginine in asubject in need thereof, comprising: identifying a subject in need ofenhanced absorption of arginine, and administering to the subject aneffective amount of an isolated astragaloside compound for facilitatingtransportation of arginine across gut cells of the subject, wherein thesubject is a juvenile, an athlete, an over-weight patient, or a patienthaving a cardiovascular disease or erectile dysfunction.
 29. The methodof claim 28, wherein the astragaloside compound is an astragalosidecompound of Formula (A):

wherein R₁ is selected from the group consisting of H, OH, O-acetyl,O-xylopyranosyl, O-(2-acetylxylopyranosyl), O-(3-acetylxylopyranosyl),O-(2,3-diacetylxylopyranosyl), O-(2,4-diacetylxylopyranosyl),O-xylopyranosyl-(1-2)-β-D-glucopyranosyl, andO-xylopyranosyl-(1-2)-α-arabinopyranosyl; R₂ is selected from the groupconsisting of H, OH, O-acetyl, O-glucopyranosyl, and O-xylopyranosyl; R₃is selected from the group consisting of H, OH, and O-acetyl; and R₄ isselected from the group consisting of


30. The method of claim 2, wherein the astragaloside compound isselected from the group consisting of astragaloside I of Formula I:

astragaloside II of Formula II:

astragaloside III of Formula III:

astragaloside IV of Formula IV:

isoastragaloside I of Formula V:

astragaloside VI of Formula VI:

isoastragaloside II of Formula VII:

cycloastragenol-6-O-β-D-glucopyranose of Formula VIII